Method of regulating a condition of mammalian keratinous tissue

ABSTRACT

A method of regulating mammalian keratinous tissue that includes applying a safe and effective amount of achachariu to a target portion of keratinous tissue. The achachariu can be in the form of a bioactive ingredient that exhibits anti-inflammatory, anti-oxidant, anti-aging, and/or anti-protein loss properties, and which is free or substantially free of benzophenones and/or protein. The achachariu ingredient may be a serum fraction and/or an extract, and may be incorporated into a personal care composition formulated for topical use on skin and/or hair.

FIELD

The present invention relates generally to regulating a condition ofmammalian keratinous tissue with achachariu. In some instances,achachariu serum fractions and/or extracts are used to regulate a skinor hair condition, for example, by incorporating the fraction or extractinto a suitable personal care composition.

BACKGROUND

Mammalian keratinous tissue, particularly human skin and hair, isconstantly subjected to a variety of insults from both extrinsic andintrinsic sources. Extrinsic sources include ultraviolet radiation,environmental pollution, wind, heat, infrared radiation, low humidity,harsh surfactants, abrasives, and the like, whereas intrinsic sourcesinclude chronological aging and other biochemical changes from withinthe body. Whether from an extrinsic or intrinsic source, these insultscan lead to visible signs of damage to skin and hair. In skin, thisdamage may manifest as undesirable thinning, fine lines, wrinkling,hyperpigmentation, sallowness, sagging, dark under-eye circles, puffyeyes, enlarged pores, diminished rate of skin cell turnover, flaking,scaling, dryness, and/or roughness. For hair, these insults cancontribute to, for example, hair bleaching, split ends, fragility,roughness, hair loss, reduction in hair growth rate. Currently, thereare a number of personal care products available to consumers forimproving the health and physical appearance of keratinous tissues, themajority of which are directed to delaying, minimizing or even reversingthe changes typically associated with aging or environmental damage.However, there is a continuing need for products and methods that seekto remedy these undesirable keratinous tissue conditions.

One potential source of bioactive ingredients for regulating conditionsin mammalian keratinous tissue is achachariu. Achachariu (Garciniahumilis) is a plant belonging to the Clusiaceae (or Guttiferae) familyand is widely distributed in the region of Santa Cruz, Bolivia. Theachachariu plant and/or fruit is used in Bolivian folk medicine for itshealing, digestive, and laxative properties. In Brazil, achachariu ispopularly known as “achacha” and is used in folk medicine to treatrheumatism, inflammation, pain and gastric disorders (Alves T M A, SilvaA F, Brandao M, Grandi T S M, Smânia E F et al. (2000) “BiologicalScreening of Brazilian Medicinal Plants.” Mem Inst Oswaldo Cruz 95:367-373 and Barbosa W, Chagas E A, Martins L, Pio R, Tucci M L et al.(2008) “Germinação de sementes e desenvolvimento inicial de plantulas deachachariu.” Rev Bras Frutic 30: 263-266).

Some studies suggest that achachariu, like many plants, containsunwanted components of concern, such as benzophenones (e.g., guttiferoneA) and proteins. See, e.g., Acuña U M, et al., (2009) “Polyisoprenylatedbenzophenones from Clusiaceae: potential drugs and lead compounds.” CurrTop Med Chem 9: 1560-1580). Benzophenones are known to exhibit variousbiological activities such as cytotoxic, genotoxic antimicrobial,antiviral and antioxidant activity. See, e.g., Terrazas P M, et al.,(2013) “Benzophenone guttiferone A from Garcinia achachairu Rusby(Clusiaceae) Presents Genotoxic Effects in Different Cells of Mice.”PLoS ONE 8(11): e76485). And proteins are known to cause allergicreactions when contacted with skin. See, e.g., V. Janssens, et al.,(2015) “Protein contact dermatitis: myth or reality?” British Journal ofDermatology; 132: 1-6). Thus, in some instances it may be desirable toremove these unwanted components or reduce their concentrationssubstantially when providing an achachariu ingredient for incorporationinto a personal care composition.

SUMMARY

Disclosed herein is a method of regulating a condition of mammaliankeratinous tissue, comprising: identifying a target portion ofkeratinous tissue in need of treatment or where treatment is desired;and applying a personal care composition comprising an effective amountof an achachariu serum fraction to the target portion of keratinoustissue. The method may include one or more of the following features inany combination: the achachariu serum fraction is obtained fromachachariu fruit; the achachariu serum fraction is obtained from thepeel of the achachariu fruit; the achachariu serum fraction is made by aprocess comprising filtering achachariu cell juice to removeparticulates; the achachariu serum fraction has a dry matter content ofless than 25%; the personal care composition is applied to the targetportion of keratinous tissue at least once per day during a treatmentperiod; the personal care composition is a rinse-off type composition,and at least some of the personal care composition is removed from thetarget portion of keratinous tissue after application; rinsing at leastsome of the personal care composition off the target portion ofkeratinous tissue with water; removing at least some of the personalcare composition from the target portion of keratinous tissue with animplement; the achachariu serum fraction is present in the personal carecomposition at an amount of from about 0.01% to about 15%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a cross-sectional view of an achachariu fruit.

FIG. 2 is a schematic of a process for preparing bioactive serumfractions and bioactive fraction extracts derived from achachariu.

FIGS. 3A, 3B, 4A, 4B are graphs illustrating absorbance spectra ofachacha serum fractions and extracts.

FIGS. 5-8 illustrate the respective amino acid losses for serine,glutamine/glutamic acid, valine, and proline observed in a protein lossassay.

FIG. 9 illustrates the total amino acid loss observed in a protein lossassay.

DETAILED DESCRIPTION

It has now been discovered that achachariu (Garcinia humilis),especially bioactive extracts and serum fractions obtained fromparticular portions of the achachariu plant, possess properties that maybe beneficial for regulating conditions of mammalian keratinous tissue.In particular, bioactive achachariu fruit extracts and serum fractionsexhibit surprising properties related to anti-oxidation, inhibitingmelanin production, inhibiting lipogenesis, and reducing hair proteinloss, which can be exploited to provide a wide variety of different skinand/or hair health benefits. Thus, it is now possible to regulate aparticular skin and/or hair condition by applying an effective amount ofachachariu to a target portion of keratinous tissue where treatment isneeded or desired.

It may also be desirable to provide achachariu serum fractions and/orextracts that are free or substantially free of benzonphenones (e.g.,guttiferone A) and/or proteins. As used herein, “substantially free ofproteins” means less than 0.15% total protein content determined byhydrolyzed and un-hydrolyzed amino acid analysis conducted on an HitachiL-8900 amino acid analyzer, and “substantially free of benzophenones”means less than 0.1% total benzophenones, including guttiferone A, asdetermined by the method described in Dal Molin M M, et al. (2012)Phytochemical analysis and antinociceptive properties of Garciniaachachariu Rusby (Clusiaceae) seeds. Arch Pharm Res 35: 623-631.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” are intended to include the plural forms as well,unless the context clearly indicates otherwise. All percentagesdisclosed herein are by weight of the total composition, unlessspecifically stated otherwise. All ratios are weight ratios, unlessspecifically stated otherwise. The number of significant digits conveysneither a limitation on the indicated amounts nor on the accuracy of themeasurements. All numerical amounts are understood to be modified by theword “about” unless otherwise specifically indicated. All measurementsare understood to be made at 25° C. and at ambient conditions, where“ambient conditions” means conditions under about one atmosphere ofpressure and at about 50% relative humidity. All such weights as theypertain to listed ingredients are based on the active level and do notinclude carriers or by-products that may be included in commerciallyavailable materials, unless otherwise specified. All numeric ranges areinclusive of narrower ranges and combinable; delineated upper and lowerrange limits are interchangeable to create further ranges not explicitlydelineated.

The compositions of the present invention can comprise, consistessentially of, or consist of, the essential components as well asoptional ingredients described herein. As used herein, “consistingessentially of” means that the composition or component may includeadditional ingredients, but only if the additional ingredients do notmaterially alter the basic and novel characteristics of the claimedcompositions or methods.

DEFINITIONS

“About,” as used herein, modifies a particular value, by referring to arange equal to the particular value, plus or minus twenty percent(+/−20%) or less (e.g., less thanb 15%, 10%, or even less than 5%).

“Actives” means compounds that, when applied to keratinous tissue and/ora target portion of keratinous tissue, provide a benefit or improvementto the keratinous tissue. The actives herein can be skin care actives,hair care actives, or a combination thereof.

“Apply” or “application”, as used in reference to a composition, meansto apply or spread the compositions of the present invention onto ahuman skin surface such as the epidermis.

“Derivative” means a molecule similar to that of another one, butdiffering from it with respect to a certain functional moiety (e.g.,esters, ethers, amides, amines, carboxylic acids, hydroxyls, acetyls,thiols, halogens, thiols, and/or salt derivatives of the relevantmolecule).

“Dermatologically acceptable” means that the compositions or componentsdescribed are suitable for use in contact with human keratinous tissuewithout undue toxicity, incompatibility, instability, allergic response,and the like.

“Effective amount” means an amount of a compound or compositionsufficient to significantly induce a positive benefit to keratinoustissue, such as a health, appearance, and/or feel benefit, including,independently or in combination, the benefits disclosed herein, but lowenough to avoid serious side effects (i.e., to provide a reasonablebenefit to risk ratio, within the scope of sound judgment of the skilledartisan). An “effective amount of achachariu” is an amount of achachariusufficient to regulate a desired condition of mammalian keratinoustissue when topically applied thereto in a personal care composition.

“Exogenous solvent” means any solvent placed in contact with the plantmaterial for the purpose of separating compounds from the plantmaterial. It may be a solvent originally present in plant material butadded in higher amounts (e.g., in case of water extracts), or a solventnot inherently present in plant material. Plant material herein caninclude any solid or liquid portion of the AchachariuAchachariu plant.

“Extract,” when referring to achachariu, means a material (e.g., one ormore phytochemicals) obtained from a portion of achachariu plantmaterial (e.g., peel, fruit, seed, stem, bark, leaves, roots, and/or acombination of these), which may be fresh, dried, or partially dried, bycontacting the plant portion(s) with an exogenous solvent and separatingthe desired material from the solvent using a conventional extractionprocess. Conventional extraction processes for obtaining plant extractsare well known in the art.

“Hair care composition” refers to a composition for regulating and/orimproving a hair condition. Some nonlimiting examples of hair-carecompositions include shampoos and hair conditioners.

“Hair conditioning agent” includes cationic surfactants, high meltingpoint fatty compounds, a silicone compounds, and mixtures thereof thatprovide a conditioning benefit to hair. Hair care compositions thatinclude a hair conditioning agent may be referred to as “hairconditioners.” However, it is to be appreciated that shampoos may alsoinclude a hair conditioning agent.

“Keratinous tissue” refers to keratin-containing layers positioned asthe outermost protective covering of mammals which includes, but is notlimited to, skin, hair, nails, cuticles, etc.

“Regulating the condition of mammalian keratinous tissue,” as usedherein, means improving the appearance and/or feel of keratinous tissue.

“Salts” refer to ionic forms of a given compound combined withcounterions deemed acceptable for a given application (e.g., food,topical, pharmaceutical). Examples include but are not limited tosodium, potassium, calcium, ammonium, manganese, copper, and/ormagnesium salts of a given compound.

“Shampoo” refers to a hair care composition that includes a combinationof a detersive surfactant and a dermatologically acceptable carrier.

“Shave prep composition” refers to a topical personal care compositionthat is applied before, during, and/or after shaving. “Shaving” refersto the removal of facial or body hair with a razor or similar device.Examples of shave prep compositions include shaving creams, gels, andfoams.

“Topical” refers to a composition that is intended to be applied to abodily surface such as skin or hair.

Personal Care Composition

The personal care compositions suitable for use in the methods hereininclude a safe and effective amount of achachariu (Garcinia humilis).The achachariu may be in form of a serum fraction, an extract, a powder,a juice, or a combination thereof. The achachariu may be obtained fromany part of the Garcinia humilis plant, as desired, but it may beparticularly desirable to use the achachariu fruit or a portion thereof(e.g., peel, pulp, and/or seed). Some testing suggests that theachachariu fruit, especially the peel, may contain higher concentrationsof biologically active compounds (e.g., carbohydrates, citric acid,flavonoids, xanthones and tannins) than other parts of the achachariuplant for regulating particular conditions of mammalian keratinoustissue. The achachariu extract and/or serum fraction may be obtainedaccording to the methods described in more detail below and combinedwith a dermatologically acceptable carrier, along with any optionalingredients, using conventional methods of making personal carecompositions of the type. The personal care composition may then bepackaged for commercial distribution.

FIG. 1 illustrates the distinct layers and parts of the achachariufruit, as shown by a section passing through a long axis of the fruit.Whole fruit, a particular part of fruit, or any combination of parts offruit may be used to provide a serum fraction or extract herein.However, this is not meant to exclude further distinctions of fruitparts than described here, or leaves that may be retained duringharvesting. As shown in FIG. 1, the outermost layer of the achachariufruit is the “Peel”, which is comparatively thick and tough. The nextinward layer is the “Flesh”, which is comparatively soft and yielding.The flesh surrounds a comparatively large “Seed”. Especially large fruitmay have more than one seed.

The amount of achachariu that is “effective” can differ from oneparticular source (e.g., manufacturer) to another, and can be determinedby the skilled artisan based upon a particular product's level ofactivity (e.g., level of active components present). The concentrationof active components in the particular achachariu product to be usedwill depend on factors such as the final dilution volume of the product,the particular method employed, the natural range of variation amongindividual plants, and other common factors known to those skilled inthe art. In some instances, an effective amount of achachariu may rangefrom 0.01% to 15% (e.g., from 0.1% to 10%, 0.2% to 7%, 0.5% to 5%, oreven 1% to 3%).

The personal care compositions herein are intended for topicalapplication to mammalian keratinous tissue. The present compositions maybe leave-on-type compositions such as moisturizing lotions, serums, andcreams, or rinse-off-type compositions such as body washes, shampoos,hair conditioners, shower gels, skin cleansers, cleansing milks, andshave prep compositions. Rinse-off-type compositions are appliedtopically to the skin or hair and then subsequently removed (e.g., viawashing and/or with an implement such as a towel or razor), usuallywithin minutes of application. The personal care compositions herein maybe provided in a variety of forms, including, but not limited to,emulsions, lotions, milks, liquids, solids, creams, gels, mousses,ointments, pastes, serums, sticks, sprays, tonics, aerosols, foams,and/or pencils. In some instances, the present compositions may be in aform suitable for use with an implement such as, for example, a cosmeticapplicator (powered or unpowered); a reusable or disposable wipe,tissue, towel, or diaper; a razor or other shaving device; a personalcleansing implement such as a mesh shower sponge, conventional sponge,or wash cloth; a swab; and/or a pen.

FIG. 2 illustrates an exemplary method for providing bioactive achachriuingredients. It is to be appreciated that portions of the processreferring to “fruit” can include any portion of the achachariu plant, asdesired. The individual steps illustrated in FIG. 2 are described inmore detail below.

“Cleaning” involves the removal of debris from the harvested achachariuplant material, prior to further processing, in a way that avoids injuryto the fruit or removal of juice or other desirable components. Forexample, cleaning can be performed by low-pressure rinsing with potablewater, under conditions where residual water wash would not noticeablycontain plant pigments.

“Selection of material” involves separating various parts of theachachariu plant material to further process a particular part alone, orin combination with other parts. This includes, but is not limited toselecting whole fruit, peel, flesh, seed and/or leaves for furtherprocessing.

“Maceration” is an optional step for rendering the plant material and/orselected parts of the plant material into smaller particles andotherwise disrupting the integrity of the fruit to ease the followingexpelling of liquid juice. If the selected plant material is especiallyyielding (such as the flesh of the fruit) or otherwise deemed suitablefor conditions and equipment of the pressing step, maceration may beomitted. Examples of suitable maceration implements include, but are notlimited to, devices such as a crusher, a grinder, or a mill (e.g., knifemill or hammer mill). To prevent temperature-induced degradation ofplant material, maceration can include temperature monitoring andselection of maceration parameters which minimize the risk oftemperature increase during this step.

“Pressing” involves the application of mechanical force to the selected,and optionally macerated, achachariu plant material to separate at leastsome of the liquid component from the rest of the plant material. Theapplied mechanical force may be provided by any suitable means known inthe art (e.g., ambient gravity, centrifugal force from a rotaryexpeller, pressure from the piston of a hydraulic press, or rollers or ascrew of appropriate type of press).

“Mechanical separation” involves the removal of relatively largeachachariu particles (e.g., pulp or agglomerated/aggregated cloudparticles) from the liquid component of the achachariu plant material.

“Juice” refers to the liquid material expelled from achachariu plantmaterial as a result of pressing. Juice can contain solid particles,semi-solid particles, and/or droplets of water-immiscible liquids of avariety of sizes (collectively referred to as “achacaha particles”) inan aqueous serum. Achachariu particles, based on size and ease ofremoval, can be qualitatively described as either “pulp” or “cloud”.While details depend on properties of the involved materials (e.g. wholefruit versus selected parts) and exact processing parameters, an exampleof a size boundary between “pulp” and “cloud” could lie between about 1and about 100 microns.

“Pulp” refers to relatively large achachariu particles present in juiceand those same achachariu particles removed from the juice by mechanicalseparation. It is not uncommon to see and distinguish individual pulpparticles with a naked eye. Pulp particles suspended in the juice areamenable to removal by mechanical separation, including, but not limitedto, sedimentation by ambient gravity, skimming, passing through a meshor a filter, or centrifugation.

“Cloud” refers to relatively small achachariu particles present in theachachariu juice, and may even include dissolved compounds (e.g., highmolecular weight compounds such as proteins and polysaccharides), whichcan be readily induced (e.g., by coagulation or temperature change) toform particles. Cloud particles are typically visible as turbidity ofthe achachariu juice, and individual cloud particles are generallyindistinguishable to the naked eye. Cloud particles are generallydispersed in the achachariu juice and such dispersions tends to remainstable for a longer period time than a pulp suspension, especiallycolloidal components of the cloud. Removal of cloud particles bymechanical means such as those used for pulp removal can be difficult.

“Destabilizing treatment” generally involves exposing a material toelectromagnetic waves in order to modify one or more physical propertiesof the material (e.g., the real component of low-frequency dielectricconstant). With regard to achachariu juice, destabilization treatmentdegrades stability of the particle dispersion in the juice by causingagglomeration and/or aggregation of particles (especially cloudparticles) into assemblies that are sufficiently large and stable toenable and/or improve their subsequent removal by mechanical separationtechniques such as those described above for pulp removal.

“Serum” refers to liquid portion of achachariu juice remaining afterpulp and cloud, as well as possible contaminants of concern, has beenremoved. Achachariu serum is free or substantially free of achachariuparticles.

“Finished ingredient” refers to serum, extract, and/or particle-freeextract that is in a form suitable for subsequent sale and/orincorporation into a personal care product. The finished ingredient mayinclude preservatives that protect the serum, extract, particle-freeextract, and/or compositions containing the same against environmentalchallenges such as temperature, atmosphere (e.g., oxygen), light, andmicroorganisms. It is to be appreciated that an ingredient free ofpreservatives is also contemplated herein. Surprisingly, it has beenfound that the processes described herein produce finished ingredientswith multifunctional biological activities that are either free of orsubstantially free of benzophenones and proteins.

“Post-destabilization precipitate” refers to achachariu particlesremoved from the juice via mechanical separation following destabilizingtreatment.

“Solvent extraction” in FIG. 2 involves conducting an extraction onachachariu particles removed from the achachariu juice with one or moresolvents (e.g., dipropylene glycol).

The extract shown in FIG. 2 refers to the solution resulting from thesolvent extraction of the achachariu pulp and post-destablizationprecipitate. In some instances, any achachariu particles remaining inthe extract may be separated (e.g., mechanically) to yield aparticle-free extract. The extract and/or particle-free extract mayserve as a finished ingredient, or as a base for a finished ingredientvia addition of preservatives.

“Post-extraction precipitate” refers to achachariu particles separatedfrom the extract in FIG. 2.

Achachariu Serum Fraction

The compositions herein may include an achachariu serum fraction and/orsalt, isomer, or derivative thereof. Serum fraction techniques involveseparating the fresh cell juice found in plant material from the rest ofthe plant matter and processing the juice to provide a stable, refinedserum. Examples of achachariu serum fractions that may be suitable foruse in the compositions and methods herein include Recentia® GH (wholefruit) and Recentia® GH-P (peel only) from Ashland SpecialtyIngredients. Achachariu peel serum fraction has the INCI designation“Garcinia Humilis Peel Extract” and the CAS No. 1622986-60-0.

The achachariu serum fractions herein may be prepared from freshachachariu using a method that helps maintain the integrity of thebioactive components present in the achachariu plant material. Careshould be taken to preserve the achachariu plant material integrityduring harvesting and transport, so as to minimize environmental factorssuch as moisture loss and biological degradation. All steps of the serumfraction making process should be completed in the shortest possibleperiod of time to minimize exposure of the fresh achachariu plantmaterial to sun, high temperature, and other undesirable environmentalfactors. Harvesting (e.g., by hand or mechanical cutting) should beconducted in a manner that avoids or minimizes the chopping, mashing,crushing, or other type of injury to the achachariu plant material to beused (e.g., fruit). Harvest and transport of achachariu plant materialshould be conducted in a manner to avoid moisture loss or spoilage.After harvesting, the achachariu plant material (e.g., fruit, leaves) iscleaned to remove debris prior to further processing. Cleaning isperformed under conditions to prevent the initiation of the release ofthe juice from the fruit, to cause injury, or to remove valuablecomponents. For example, the harvested achachariu plant material may bewashed with water at low-pressure (e.g., 1 kg/cm² or less) for a shortduration (e.g., 5 minutes or less). Excess water is removed from washedfruit biomass before processing.

The washed plant material is processed (e.g., macerated and pressed) toyield juice. In some instances, the whole fruit or a portion thereof(e.g., peel, fruit flesh, and/or seed) is macerated (e.g., by grinding)and then pressed to separate the juice from the rest of the plantmatter. For example, a hammer mill may be used to grind the whole fruitto yield plant material particles of a desired size (e.g., less than orequal to 0.5 cm) in a short time (e.g., 10 seconds or less) and withoutsignificant increase of biomass temperature (e.g., 5° C. or less). Theseparation of juice from the achachariu plant material is commenced assoon as possible after maceration of the achachariu plant to avoid asignificant increase in temperature of the juice and/or pulp. Forexample, macerated achachariu leaves can be pressed using a horizontal,continuous screw press, wherein the pressure on the cone is maintainedat 24 kg/cm², screw speed is at 12 rpm, and biomass temperature increaseis 5° C. or less.

The achachariu juice can be subjected to further processing immediately,or the juice may be frozen at about −30° C. and thawed and processedlater. Achachariu juice typically contains a variety of achachariuparticles (i.e., pulp and cloud) in an aqueous serum. Thus, in order toobtain the desired serum fraction, all or substantially all of theachachariu particles should be removed from the juice. In someinstances, it may be desirable to provide a serum fraction that includesless than 25% dry matter (e.g., less than 20%, 15%, 12%, 10% or evenless than 9% dry matter). The pulp may be removed from the achachariujuice using a suitable mechanical separation technique such as, forexample, straining, filtration (including filtration utilizing apressure gradient), skimming, sedimentation by ambient gravity,decanting, centrifugation, and/or a combination of these to yieldpulp-free or substantially pulp-free juice. The cloud may be removed byexposing the juice to electromagnetic waves to destabilize the clouddispersion in the juice. The frequency of electromagnetic waves mayrange between 300 MHz and 50 GHz. Suitable devices for generating suchelectromagnetic waves include, but are not limited to magnetrons, powergrid tubes, klystrons, klystrodes, crossed-field amplifiers, travellingwave tubes, and gyrotrons. In some instances, a magnetron operating at afrequency of 915 MHz, 2.45 GHz, and 5.8 GHz can allow the value of realcomponent of low-frequency dielectric constant (∈^(′) ₀) to be decreasedduring the treatment by between 10 and 40 compared to its value prior totreatment. The value of real component of low-frequency dielectricconstant (∈^(′) ₀) can be determined using broadband dielectricspectroscopy data obtained via equipment and software from AgilentTechnologies: PNA-L Network Analyzer N5230C with 85070E dielectric probekit, N4693-60001 electronic calibration module, and 85070 software. Thecalculation is performed according to method described in the articleCole, K. S., & Cole, R. H. (1941). Dispersion and absorption indielectrics I. Alternating current characteristics. The Journal ofChemical Physics, 9(4), 341-351.

Other non-limiting examples of techniques for providing serum fractionsare disclosed in U.S. Pat. Nos. 7,442,391 and 7,537,791 to Koganov andU.S. Pub. No. 2012/0201768, filed by Swanson, et al.

Achachariu Extract

The compositions and methods herein may employ an achachariu extract. Anachachariu extract is obtained by separating compounds from achachariuplant matter with an exogenous solvent. Consistent with the generalprinciple of “like dissolves like,” the choice of extraction solventlargely determines the type and number of compounds that will resultfrom any particular extraction technique. For instance, polar compoundsare typically extracted out by using a polar solvent, while non-polarcompounds are extracted out by using a non-polar solvent. Thecorrelation between solvent polarity and the types of materials isolatedusing traditional solvent extraction is described in Houghton & Raman,Laboratory Handbook for the Fractionation of Natural Extracts (1998).

The achachariu extracts herein may be obtained using any suitableextraction technique known in the art. In some instances, the achachariuextract may be obtained by the following procedure: (i) place thedesired portion of dried plant material (e.g., whole fruit, fruit pulp,peel, seeds, stem, bark, leaves) in a conical glass percolator; (ii) addthe indicated percentage of extraction solvent in a w/w ratio of 1 partplant material to 2 parts extraction solvent (when the indicatedpercentage of extraction solvent is less than 100%, the remainingsolvent is water (e.g., 95% ethanol with 5% water, 50% ethanol with 50%water)); (iii) allow the extraction to proceed for 16 to 24 hours; (iv)collect the percolate, and repeat the above process until the resultingpercolate is substantially free from plant additional extract; (v)combine the percolates, evaporate to dryness under reduced pressure, andstore the resulting extract under nitrogen at less than 4 degreesCelsius. Extracts may be used without any further modification or may bemodified (e.g., ethoxylated, esterified) to form a derivative material.

Achachariu Powder or Juice

The personal care compositions for use with the methods herein mayinclude an achachariu powder or juice. Achachariu juice may be obtainedby macerating and process achachariu plant material, for example, asdescribed hereinabove. Achachariu powder may be obtained by dryingachachariu plant material and processing the dried material intoparticles. Additionally or alternatively, the achachariu plant materialmay be processed into particles and then dried. Processing theachachariu into particles may include one or more grinding stepsperformed before and/or after drying. The achachariu biomass may bedried and processed into particles using any suitable method known inthe art. The achachariu powder may have a weight average particle sizeof from 50 to 750 microns. Once the achachariu is processed intoparticles of the desired size, the powder can be incorporated into apersonal care composition using conventional methods for making suchcompositions.

Dermatologically Acceptable Carrier

The personal care compositions for use with the methods herein include adermatologically acceptable carrier at an amount of 20% to 99.99% (e.g.,50% to 99%, 60% to 98%, 70% to 95%, or even 60% to 80%) by weight of thecomposition. The carrier may be aqueous or anhydrous. The form of thecarrier is not particularly limited, and can be any suitable form knownin the art for the application desired (e.g., solutions, dispersions,emulsions and combinations thereof). “Emulsions” refer to compositionshaving an aqueous phase and an oil phase. Emulsion carriers include, butare not limited to oil-in-water, water-in-oil and water-in-oil-in-wateremulsions. Emulsion carriers herein may include from 0.01% to 10% (e.g.,0.1% to 5%) of an emulsifier (e.g., nonionic, anionic, cationicemulsifier, or a combination thereof). Suitable emulsifiers aredisclosed in, for example, U.S. Pat. No. 3,755,560, U.S. Pat. No.4,421,769, and McCutcheon's Detergents and Emulsifiers, North AmericanEdition, pages 317-324 (1986).

Optional Ingredients

The present compositions may contain a variety of optional ingredientsthat are conventionally used in the particular product type provided, aslong as they do not undesirably alter product stability, aesthetics, orperformance. The optional ingredients, when incorporated into thecomposition, should be suitable for use in contact with human keratinoustissue without undue toxicity, incompatibility, instability, allergicresponse, and the like within the scope of sound judgment. The CTFACosmetic Ingredient Handbook, Second Edition (1992) describes a widevariety of nonlimiting cosmetic and pharmaceutical ingredients. Thecompositions herein may include 0.0001% to 50% (e.g., 0.001% to 20% oreven 0.01% to 10%), by weight of the composition, of optionalingredients. Some non-limiting examples of optional ingredients includeabrasives, absorbents, aesthetic components such as pigments, opacifyingagents, colorings/colorants, particles, essential oils, anti-cakingagents, foaming agents, anti-foaming agents, binders, biologicaladditives, vitamins, minerals, moisturizers, emollients, humectants,skin tone agents, lubricating agents, sensates (e.g., menthol),fragrances, anti-dandruff agents, buffering agents, bulking agents,chelating agents, chemical additives, biocides, denaturants,astringents, external analgesics, anti-inflammatory agents, sunscreenagents, film formers and/or polymers for aiding film-forming propertiesand substantivity of the composition, pH adjusters, propellants,reducing agents, sequestrants, conditioning agents, thickeners, andcombinations of these. Some of these and other optional ingredients aredescribed in more detail below.

The personal care compositions herein may include 0.1% to 10% (e.g.,0.5% to 7%) of a conditioning agent to provide a particular conditioningbenefit to hair and/or skin. For example, some suitable hairconditioning agents include silicones (e.g., silicone oils, cationicsilicones, silicone gums, high refractive silicones, and siliconeresins), organic conditioning oils (e.g., hydrocarbon oils, polyolefins,and fatty esters), and combinations thereof. Some non-limiting examplesof suitable silicone conditioning agents, and optional suspending agentsfor the silicone, are described in U.S. Reissue Pat. No. 34,584; andU.S. Pat. Nos. 4,152,416, 4,364,837, 5,104,646, 5,106,609, 5,674,478,5,750,122, 7,465,439, 7,041,767, and 7,217,777. Some non-limitingexamples of skin conditioning agents include vitamins, minerals,peptides and peptide derivatives, sugar amines, sunscreens, oil controlagents, particulates, flavonoid compounds, hair growth regulators,anti-oxidants and/or anti-oxidant precursors, preservatives, proteaseinhibitors, tyrosinase inhibitors, anti-inflammatory agents,moisturizing agents, exfoliating agents, skin lightening agents, sunlesstanning agents, lubricants, anti-acne actives, anti-cellulite actives,chelating agents, anti-wrinkle actives, anti-atrophy actives,phytosterols and/or plant hormones, N-acyl amino acid compounds,antimicrobials, and antifungals. Other non-limiting examples of skinconditioning agents can be found in U.S. Pub. Nos. 2010/0272667 and2008/0206373. In addition to providing a skin health and/or appearancebenefit, skin conditioning agents for use in shave prep compositions mayalso help to reduce the coefficient of friction for the shave prepcomposition, thereby decreasing the potential for skin irritationrelated to shaving.

The personal care compositions herein may include 0.05% to 5% (e.g.,0.1% to 4% or even 0.25% to 3%) of a thickening agent. Suitable classesof thickening agents include, but are not limited to carboxylic acidpolymers, crosslinked polyacrylate polymers, polyacrylamide polymers,polysaccharides, gums and mixtures thereof. Non-limiting examples ofsuitable thickening agents are described in the CTFA InternationalCosmetic Ingredient Dictionary, 10th Ed. (2004), pp. 2294-96.

The personal care composition herein may include from 5% to 50% of ananionic, zwitterionic, and/or amphoteric detersive surfactant. Theconcentration of the detersive surfactant in the composition should besufficient to provide the desired cleaning and lather performance Somenon-limiting examples of anionic detersive surfactants include disodiumN-octadecylsulfosuccinnate; disodium lauryl sulfosuccinate; diammoniumlauryl sulfosuccinate; tetrasodiumN-(1,2-dicarboxyethyl)-N-octadecylsulfosuccinnate; diamyl ester ofsodium sulfosuccinic acid; dihexyl ester of sodium sulfosuccinic acid;dioctyl esters of sodium sulfosuccinic acid; and olefin sulfonates. Someparticularly suitable examples of anionic surfactants are alkyl andalkyl ether sulfates. Other non-limiting examples of anionic detersivesurfactants are described in U.S. Pat. Nos. 2,486,921; 2,486,922;2,396,278; and 3,332,880.

The personal care compositions herein may include a cationic surfactantsystem that includes one or more cationic surfactants. If present, thecationic surfactant system may be incorporated into the composition at alevel of 0.1% to 10% (e.g., 0.5% to 8%, 1% to 5%, or even 1.4% to 4%).In certain hair care compositions, the cationic surfactant may bebalanced to provide desirable ease-to-rinse, feel, rheology, and wetconditioning benefits.

The personal care compositions herein may include a high melting pointfatty compound. The high melting point fatty compounds useful hereintypically have a melting point of 25° C. or higher, and may be selectedfrom the group consisting of fatty alcohols, fatty acids, fatty alcoholderivatives, fatty acid derivatives, and mixtures thereof. It is to beappreciated that the compounds disclosed in this section of thespecification can in some instances fall into more than oneclassification, e.g., some fatty alcohol derivatives can also beclassified as fatty acid derivatives. However, a given classification isnot intended to be a limitation on that particular compound, but is doneso for convenience of classification and nomenclature. Further, it is tobe appreciated that, depending on the number and position of doublebonds, and length and position of the branches, certain compounds havingcertain required carbon atoms may have a melting point of less than 25°C. Such compounds of low melting point are not intended to be includedin this section. Nonlimiting examples of the high melting pointcompounds are found in International Cosmetic Ingredient Dictionary,Fifth Edition, 1993, and CTFA Cosmetic Ingredient Handbook, SecondEdition, 1992.

The personal care compositions herein may include a cationic and/or anon-ionic polymer at an amount of 0.05% to 3% (e.g., 0.075% to 2.0%, oreven 0.1% to 1.0%). Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm (e.g., at least 0.9 meq/gm, 1.2 meq/gm,or even at least 1.5 meq/gm), but typically less than 7 meq/gm or evenless than 5 meq/gm, at the pH of intended use of the composition, whichmay range from pH 3 to pH 9. Herein, “cationic charge density” of apolymer refers to the ratio of the number of positive charges on thepolymer to the molecular weight of the polymer. The average molecularweight of such suitable cationic polymers will generally be between10,000 and 10 million, (e.g., between 50,000 and 5 million or evenbetween 100,000 and 3 million). Some non-limiting examples of suitablecationic polymers include copolymers of vinyl monomers having cationicprotonated amine or quaternary ammonium functionalities with watersoluble spacer monomers such as acrylamide, methacrylamide, alkyl anddialkyl acrylamides, alkyl and dialkyl methacrylamides, alkyl acrylate,alkyl methacrylate, vinyl caprolactone, vinyl pyrrolidone, and cationicguar gum derivatives such as guar hydroxypropyltrimonium chloride. Somenon-limiting examples of non-ionic polymers are polyalkylene glycolshaving a molecular weight of more than about 1000. Other non-limitingexamples of polymers are disclosed in the CTFA Cosmetic IngredientDictionary, 3rd edition, edited by Estrin, Crosley, and Haynes, (TheCosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C.(1982)).

The personal care compositions herein may include a suspending agent atconcentrations effective for suspending water-insoluble material indispersed form in the compositions or for modifying the viscosity of thecomposition. Such concentrations range from 0.1% to 10%, or even from0.3% to 5.0%. Suspending agents useful herein include anionic polymers,nonionic polymers, and vinyl polymers.

The personal care compositions herein may include particles. Theparticles may be dispersed water-insoluble particles. The particles maybe inorganic, synthetic, or semi-synthetic. The particles may have amean average particle size of between 1 μm and 1 mm (e.g., between 10 μmand 800 μm, 50 μm and 500 μm, or even between 100 μm and 400 μm.

In some instances, the above cationic surfactants, together with highmelting point fatty compounds and an aqueous carrier, may be combined toform a gel matrix in a hair care composition. A gel matrix may besuitable for providing certain hair conditioning benefits such asslippery feel during the application to wet hair and softness andmoisturized feel on dry hair. In view of providing the above gel matrix,the cationic surfactant and the high melting point fatty compound arecontained at a level such that the weight ratio of the cationicsurfactant to the high melting point fatty compound is in the range of,for example, 1:1 to 1:10, or even 1:1 to 1:6.

Methods of Regulating Keratinous Tissue Condition

Personal care compositions comprising an effective amount of achachariucan be used to regulate a condition of mammalian keratinous tissue bytopically applying the personal care composition herein to mammaliankeratinous tissue. The present method may include identifying a targetportion of keratinous tissue in need of treatment and/or where treatmentis desired, and then applying the personal care composition to thetarget portion of keratinous tissue. Identifying a target portion ofkeratinous tissue may be based on, for example, the presence of avisible condition (e.g., dryness, brittleness, split ends, undesirablefeel, redness, inflammation, discoloration, or wrinkles). In someinstances, the target portion of keratinous tissue may not exhibitvisible signs of a condition, but a user may still wish to target suchan area if it is one that is known to develop an undesirable condition(e.g., skin or hair surfaces that are typically not covered by clothingand areas of skin prone to shaving irritation).

Personal care compositions containing an effective amount of achachariumay be applied once a day, twice a day, or on a more frequent dailybasis, during a treatment period. The treatment period is ideally ofsufficient time for the achachariu to provide the desired benefit. Forexample, the treatment period may be of sufficient time for theachachariu to provide a noticable and/or measurable improvement in ahair or skin condition. The treatment period may last for at least 1week (e.g., about 2 weeks, 4 weeks, 8 weeks, or even 12 weeks). In someinstances, the treatment period will extend over multiple months (i.e.,3-12 months) or multiple years. In some instances, a cosmeticcomposition containing an effective amount of achachariu may be appliedmost days of the week (e.g., at least 4, 5 or 6 days a week), at leastonce a day or even twice a day during a treatment period of at least 2weeks, 4 weeks, 8 weeks, or 12 weeks.

The personal care compositions herein may be applied locally orgenerally. In reference to application of the composition, the terms“localized”, “local”, or “locally” mean that the composition isdelivered to the targeted area while minimizing delivery to keratinoussurfaces where treatment is not needed or desired. While certainembodiments herein contemplate applying a composition locally to anarea, it will be appreciated that the compositions herein can be appliedmore generally or broadly to one or more keratinous surfaces. In certainembodiments, the compositions herein may be used as part of a multi-stepbeauty regimen, wherein the present composition may be applied beforeand/or after one or more other compositions.

Using the personal care compositions herein according to the presentmethods can provide a health or appearance benefit to the treatedkeratinous tissue. Some non-limiting examples of skin benefits includereducing the appearance of wrinkles, deep lines, fine lines, crevices,bumps, large pores; increasing the convolution of the dermal-epidermalborder; skin lightening; increasing elasticity, decreasing sagging,reducing cellulite; reducing the appearance of under-eye circles,reducing the appearance of discoloration, reducing hyperpigmentation,increasing skin luminosity, and combinations thereof. Some non-limitingexamples of hair benefits include shine, softness, combability,antistatic properties, wet-handling, damage, anti-dandruff,manageability, body, and greasiness.

The personal care compositions may be applied by any suitable meansknown for applying such products, including rubbing, wiping or dabbingwith hands, fingers and/or an implement. Non-limiting examples ofimplements include a sponge or sponge-tipped applicator, a swab (forexample, a cotton-tipped swab), a pen optionally comprising a foam orsponge applicator, a loofa, a brush, a wipe, a cloth, and combinationsthereof. The composition may be pre-applied to the applicator and, forexample, delivered to the user pre-packaged as such, or the user may beinstructed to apply the composition to the applicator prior to use. Insome instances, the composition may be stored in an implement, forexample, in a separate storage area for the composition. In such anexample, the composition may be transferred to the applicator from thestorage area, for example, by squeezing, breaking, and/or other suitablemeans. The composition may be applied to the keratinous tissue bycontacting the applicator and composition to the skin. Contact mayinclude, for example, light pressure, dabbing, rubbing, wiping, and thelike. When targeted application is desired, the composition may beapplied only to the target area of keratinous tissue.

Methods of Determining the Effectiveness of Achachariu

Irritation and inflammation in human skin can lead to visible signs ofskin aging (e.g. discoloration and wrinkles), decreased mechanicalstrength, decreased protective functions, and lessened ability torecover from stress and injuries. Ubiquitous stressors such as sunlightand surfactants can be especially problematic. Mitigating irritation andinflammation of the skin, particularly those caused by common stressors,is important and desirable.

The adverse effects of light, most commonly sunlight (though artificialsources are included), on human skin are well known. Overly highexposure to sunlight may cause acute adverse reaction involvingirritation and inflammation, such as sunburn. Exposures insufficient tocause acute reactions can still trigger inflammation-related processes.Accumulated inflammatory damage from sunlight exposure causesdegradation of skin resilience and development of an undesirableappearance (i.e., photoaging).

Surfactants are also known to cause adverse reactions on human skin.Surfactants are used in a variety of personal care and cleansingproducts to allow or improve processes of cleansing, foaming,emulsifying, solubilizing, and dispersing. Repetitive contact withsurfactant-containing products has been shown to cause damage of theskin barrier due to surface or interface activities of the surfactants,which can be perceived by consumers as dryness, itchiness, swelling,redness, and/or pain. The weakened barrier subsequently leads to deeperpenetration of the surfactants into skin and induced irritation andinflammation. As surfactants are widely used in hand soaps, facial andbody washes, shampoos and conditioners, as well as dish, laundry andhousecleaning detergents, human skin contact with surfactants is common.

Irritation and inflammation are commonly viewed as a “cascade”proceeding from necessary release of a signaling compound Interleukin(IL) 1-alpha (or IL-1α) to induction of other downstream cytokines andchemokines such as interleukins IL-6 and IL-8 or other signalingmolecules (Weiss T, Basketter D A, Schroder K R. In vitro skinirritation: facts and future. State of the art review of mechanisms andmodels. Toxicol In Vitro 2004; 18 (3): 231-43). However, previouslypublished data suggest that “cascade” view might not be a comprehensivemodel.

It has been shown that sodium dodecyl sulfate (SDS), a single compoundcommonly used as a benchmark source of surfactant stress in both invitro and in vivo studies, can trigger different portions of theirritation and inflammation process without significantly affectingrelease of a primary cytokine such as IL-1α, depending on concentration.The complexity of irritation and inflammation response of skin cellsimplies that signaling “network” model is a more adequate analogy than asignaling “cascade” model. This indicates that mitigation of such acomplex signaling process must affect more than one pathway, such as byusing a multifunctional bioactive ingredient.

One of the methodologies for studying and quantifying irritation andinflammation includes culturing cells of the tissue most likely to comein contact with stress sources, such as viable epidermal keratinocytesfrom human skin. Human epidermal keratinocytes (HEK) have become thefocus of attention in irritant-induced skin inflammation by virtue oftheir epidermal location, importance in maintaining the integrity of thestratum corneum barrier, and the ability to produce a variety ofinflammatory mediators. Keratinocytes can release a variety ofsignalling substances (e.g., interleukins and arachidonic acidmetabolites) in response to a range of irritants, including surfactantsand sunlight. The amounts of these signalling substances present in atissue sample can be measured via techniques such as Enzyme-LinkedImmunosorbent Assay (ELISA). Bioactive substances that reduce therelease of inflammatory signalling substances may help control the signsof irritation and inflammation in human skin.

Besides signalling substances, other very important compounds inprocesses of irritation and inflammation are those that directly causethe damage. Especially notable inflammatory damage substances are freeradicals (especially reactive oxygen species) and protein-degradingenzymes (proteases). It is possible to detect the presence or measurethe activity of such damaging substances by incubating them with asubstrate they can alter or degrade. The alteration or degradation ofthe substrate can be measured directly (e.g., by loss or development ofcolor or fluorescence) or indirectly (e.g., by ELISA). In some cases,the presence or activity of damaging substances can be measured directlyor indirectly, for example, with spectroscopic techniques (e.g.,Electron Paramagnetic Resonance techniques and the use of chromogenicartificial stable free radicals like DPPH).

The tests selected for determining the effects of serum fractions andextracts of achachariu, and personal care compositions containing thesame, include inhibition of proteases (damaging compounds) innon-cell-based bioassays, inhibition of chemokines, cytokines andprostaglandins (signalling compounds) in cultured human skin cells,inhibition of a compound capable of both signalling and causing damagein cultured human cells, and measuring release or lack thereof ofsubstances indicating cell damage or sensitization. These testsillustrate safety (e.g., via a lack of cytotoxicity and sensitizationpotential) and the efficacy and potency of achachariu in inhibitingsignalling and damaging compounds involved in skin irritation,inflammation, and aging processes. The cells in the assays describedherein are normal human adult epidermal keratinocytes (HEK), unlessspecifically stated otherwise.

EXAMPLES

The following examples are provided for the purpose of illustration onlyand should in no way be construed as being limiting, but rather shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1 Preparation of a Serum Fraction from Achachariu Whole Fruit

In this example, achachariu (Garcinia humilis) whole fruit are used toproduce a serum fraction. The achachariu fruit are harvested fresh fromtrees, inspected, and cleaned prior to processing. Unsound fruit is notused. Selected whole fruit are ground, pressed, and mechanicallyseparated to provide juice and pulp. The yield of juice from achachariuwhole fruit after grinding, pressing and mechanical separation is about60 to 69% weight/weight; the pH of juice from achachariu whole fruit isfrom 3.0 to 3.3. The juice is immediately subjected to destabilizingtreatment by electromagnetic waves in a continuous flow system thatincludes magnetrons operating at a frequency of 5.8 GHz. The parametersof the electromagnetic waves in the destabilization step are set toachieve the decrease in value of real component of low-frequencydielectric constant (∈^(′) ₀) during the treatment by about 20 Faradsper meter (F/m) compared to its value prior to treatment. Thede-stabilized juice is immediately pumped through a continuous flowcentrifuge to yield a serum and post-destabilization precipitate. Theserum is substantially free of benzophenones and protein.

Example 2 Preparation of Serum Fraction from Achachariu Peel

In this example, achachariu fruit peel is used to produce a serumfraction. The achachariu fruit are harvested fresh from trees,inspected, and cleaned prior to processing. Unsound fruit is not used.The fruit peel is separated from whole fruits and then ground, pressed,and mechanically separated to produce juice and pulp. The yield of juicefrom achachariu fruit peel after grinding (maceration), pressing andmechanical separation is about 25 to 35% weight/weight; the pH of juicefrom achachariu fruit peel is 2.7 to 3.0. The juice from fruit peel isimmediately subjected to destabilizing treatment by electromagneticwaves in a continuous flow system that includes magnetrons operating ata frequency of 2.45 GHz. The parameters of the electromagnetic waves inthe destabilization step are set to achieve the decrease in value ofreal component of low-frequency dielectric constant (∈^(′) ₀) during thetreatment by about 30 Farads per meter (F/m) compared to its value priorto treatment. The de-stabilized juice is immediately pumped through acontinuous flow centrifuge to yield a serum and post-destabilizationprecipitate. The serum is free or substantially free of benzophenonesand protein.

Example 3 Preparation of Serum Fraction from Achachariu Fruit Flesh andSeeds

In this example, achachariu fruit flesh and seeds are used to produce aserum fraction. The achachariu fruit are harvested fresh from trees,inspected, and cleaned prior to processing. Unsound fruit is not used.The fruit flesh and seed are separated from whole fruits and thenground, pressed and mechanically separated to produce juice and pulp.The yield of juice from Achachariu (Garcinia humilis) fruit flesh andseeds after grinding, pressing and mechanical separation is about 30 to40% weight/weight; the pH of juice from achacha fruit flesh and seeds is3.8 to 4.2. The juice is immediately subjected to destabilizingtreatment by electromagnetic waves in a continuous flow system thatincludes magnetrons operating at a frequency of 2.45 GHz. The parametersof the electromagnetic waves in the destabilization step are set toachieve the decrease in value of real component of low-frequencydielectric constant (∈^(′) ₀) during the processing by about 25 Faradsper meter (F/m) compared to its value prior to treatment. Thede-stabilized juice is immediately pumped through a continuous flowcentrifuge to yield a serum and post-destabilization precipitate. Theserum is free or substantially free from benzophenones and protein.

Example 4 Preparation of Extracts from Achachariu Fruit by SolventExtraction

Solvent extractions were conducted with pulp particles, withpost-destabilization precipitate particles, including cloud, or withtheir combination using one or more solvents under particular extractionconditions: type of solvent(s), ratio of solvent to plant material(pulp, or post-destabilization precipitate, or their combinations),extraction temperatures, agitation, time of exposure, etc., in order toextract materials that are soluble in the particular solvents. The ratioof pulp and/or post-destabilization precipitate to solvent can rangefrom 1:2 to 1:1000. Extraction temperature can range from 4° C. to 100°C. Time of the exposure may vary from about 15 minutes to about 96 hrs.

Example 5—Personal Care Composition Formulations

Exemplary personal care compositions comprising achachariu are providedin Tables 1, 2, and 3 below.

TABLE 1 Shampoo EXAMPLE COMPOSITION I II III Ingredient % % % Water q.s.q.s. q.s. Polyquaternium 76¹ 2.50 — — Guar, Hydroxylpropyl — 0.25 —Trimonium Chloride² Polyquaterium 6³ — — 0.79 Sodium Laureth Sulfate21.43 21.43 21.43 (SLE3S)⁴ Sodium Lauryl Sulfate 20.69 20.69 20.69(SLS)⁵ Silicone⁶ 0.75 1.00 0.5 Cocoamidopropyl Betaine⁷ 3.33 3.33 3.33Cocoamide MEA⁸ 1.0 1.0 1.0 Ethylene Glycol Distearate⁹ 1.50 1.50 1.50Sodium Chloride¹⁰ 0.25 0.25 0.25 Achachariu Serum Fraction¹¹ 0.1 0.5 2.0Fragrance 0.70 0.70 0.70 Preservatives, pH adjusters Up to Up to Up to1% 1% 1% ¹Mirapol AT-1, Copolymer of Acrylamide(AM) and TRIQUAT, MW =1,000,000; CD = 1.6 meq./gram; 10% active; Rhodia ²Jaguar C500, MW -500,000, CD = 0.7; Rhodia ³Mirapol 100S, 31.5% active; Rhodia ⁴SodiumLaureth Sulfate, 28% active; P&G ⁵Sodium Lauryl Sulfate, 29% active; P&G⁶Glycidol Silicone VC2231-193C ⁷Tegobetaine F-B, 30% active; GoldschmidtChemicals ⁸Monamid CMA, 85% active; Goldschmidt Chemical ⁹EthyleneGlycol Distearate, EGDS Pure; Goldschmidt Chemical ¹⁰Sodium Chloride USP(food grade); Morton; note that salt is an adjustable ingredient, higheror lower levels may be added to achieve target viscosity. ¹¹Recentia ®GH; AkzoNobel

TABLE 2 Hair Conditioner EXAMPLE COMPOSITION I II III Ingredient % % %Water q.s. q.s. q.s. Silicone A¹ 1.0 — — Silicone B² — 0.5 — Silicone C³— — 0.5 Cyclopentasiloxane⁴ — 0.61 1.5 Behenyl trimethyl 2.25 2.25 2.25ammonium chloride⁵ Isopropyl alcohol 0.60 0.60 0.60 Cetyl alcohol⁶ 1.861.86 1.86 Stearyl alcohol⁷ 4.64 4.64 4.64 Disodium EDTA 0.13 0.13 0.13NaOH 0.01 0.01 0.01 Benzyl alcohol 0.40 0.40 0.40Methylchloroisothiazolinone/ 0.0005 0.0005 0.0005 Methylisothiazolinone⁸Panthenol⁹ 0.10 0.10 0.10 Panthenyl ethyl ether¹⁰ 0.05 0.05 0.05Achachariu Serum Fraction¹¹ 0.1 0.5 2 Fragrance 0.35 0.35 0.35 ¹GlycidolSilicone VC2231-193C ²Glycidol Silicone VC2231-193F ³Glycidol SiliconeVC2231-193A ⁴SF1202; Momentive Performance Chemicals ⁵Genamin TM KMP;Clariant ⁶Konol TM; Shin Nihon Rika ⁷Konol TM; Shin Nihon Rika ⁸KathonTM CG (Methylchloroisothiazolinone/Methylisothiazolinone); Rohm & Haas⁹Panthenol; Roche ¹⁰Panthenyl ethyl ether; Roche ¹¹Recentia ® GH-P;AkzoNobel

TABLE 3 Shave Prep Composition EXAMPLE COMPOSITION I II III Ingredient %% % Water q.s. q.s. q.s. Sepigel 305 (Polyacrylamide & 2.00 2.00 2.00C13-14 Isoparaffin & Laureth-7) Polyox N12K (PEG-23M) 0.50 0.50 0.50Natrosol 250 HHR (HEC) 0.80 0.80 0.80 Glycerin — — 0.50 Brij 35(Laureth-23, 100% 2.00 2.00 2.00 Active) Salicylic Acid — — — Arlamol E(PPG-15 Stearyl 2.00 — 2.00 Ether) Achachariu Serum Fraction⁶ 0.5  2.0 5.0  Acusol OP301 Opacifier (40% — — — solids) (Water andStyrene/Acrylic Copolymer) DC 1503 — 2.00 — Expancel 920-WE40 D24 — — —Disodium EDTA (EDETA BD) 0.10 0.10 0.10 Perfume 1 0.15 0.15 0.15 Perfume2 — — — Perfume 3 — — — Perfume 4 — — — Glydant Plus 0.20 0.20 0.20

Example 6—Preservatives

Various preservatives were tested for use with the bioactive serumfractions and bioactive extracts of achacha herein. Table 4 shows anexample of combination/concentrations of preservatives that may beparticularly suitable for preparing finished achacha ingredients (e.g.,prepared from the serum fractions described in Examples 1, 2, and 3). Ithas been found that the preservatives listed in Table 4 help prevent theformation of precipitates in a finished product.

TABLE 4 Amount Preservative (wt %) Pentylene Glycol (CAS 5343-92-0)1.90% Tetrasodium EDTA (CAS 64-02-8) 0.25% Sodium metabisulfite (CAS7681-57-4) 0.20% Potassium sorbate (CAS 590-00-1) 0.10% Sodium Benzoate(CAS 532-32-1) 0.10% Bioactive Serum Fraction or Extract 97.45%

Example 7—Physico-Chemical Characteristics

Table 5 illustrates methods for testing and evaluating certainphysico-chemical characteristics of achacaha serum fractions andextracts. It is to be appreciated that the methods are not limited tothe specific instruments or techniques shown, and equivalent instrumentsand techniques may be used to achieve substantially the same results, asknown by those skilled in the art.

TABLE 5 Property Test Method Units Appearance Determinedorganoleptically. N/A Odor Determined organoleptically. N/A ColorDetermined on Lovibond Comparator 3000 Gardner Scale. Gardner Turn oncomparator lamp. Measure 8 mL of sample into sample scale tube. Inserttube into comparator. Rotate the knobs until two color standards nearestin color to the sample have been located. Record the value of the samplecolor accordingly. If the color of the sample is substantially similarto both, rather than a single standard, then record it as a valuebetween the values of the two standards. Dry Matter Dry matter isdetermined by comparing the weights of liquid % sample with residual drymatter after water has been evaporated. Procedure is based on standardlaboratory practices commensurate with available equipment. Select asuitable aluminum weighing dish (e.g., VWR 25433-016) and weight thedish. Add approximately 4 mL of liquid sample to the dish with the dishon the scale. Determine the weight of the liquid sample by subtractingthe dish weight from the total weight. Repeat this procedure with twoadditional weighing dishes. Place the dishes in a ThermoScientific“Lindberg Blue M” Gravity oven at 105 degrees Celsius for 24 hours.After 24 hours, remove the dishes and allow them to cool forapproximately 5 minutes at room temperature. Weigh each dish. Determinethe weight of the dry sample. Calclulate dry matter percentage bydividing dry sample weight by liquid sample weight. Dry matterpercentage for the sample is the average of the dry matter percentagesfor the three dishes. Refractive index Determined using a ReichertArias ™ 500 brand refractometer nD according to instruction manualsections 6.0, 4.1 and 4.4-4.5. Temperature regulation is provided byCole-Parmer Polystat temperature controller, model number 12108-10 setat 20 C. Automatic Reading Method is enabled. Place 0.5 mL of deionizedwater on the surface of the lower measuring prism, taking care to avoidbubble formation. Close the cell and turn the shadowline adjustment knobto bring the shadowline within the crosshairs. Wait for temperature atrefractometer measuring cell to stabilize, then push Read button. Repeatuntil refractive index of deionized water is determined as 1.333 atleast three times in a row. Rinse the lower and upper surfaces of themeasuring cell with deionized water and blot dry with lint-free wipe.Place 0.5 mL of sample on the surface of the lower measuring prism.Close the cell and turn the shadowline adjustment knob to bring theshadowline within the crosshairs. Wait for temperature at refractometermeasuring cell to stabilize, then push Read button. Repeat until stablereadings have been obtained for sample material at least three times ina row. Record this value as the Refractive Index. Density Determinedwith Densito 30PX densitometer from Mettler g/cm³ Toledo. Procedure isbased on Operating Instructions for Densito 30PX, sections 4 and 6. Setthe instrument display to g/cm³. Calibrate the instrument with 4 cm³ ofdeionized water, avoiding bubble intake or formation. If the density ofthe calibration sample deviates by more than 0.05% from the expecteddensity of water at ambient temperature, recalibrate the densitometer asper Operating Instructions. Eject the calibration sample and fill thesample loops with 4 cm³ of sample, avoiding bubble intake or formation.Record the reading. Eject the sample and repeat steps above foradditional readings, until receiving three matching readings in a row.Record the value of density (specific gravity) for the sample. pHDetermined by measuring on a Denver Instrument Model 250 N/ApH/ISE/conductivity meter with pH/ATC electrode number 300729.1.Procedure is based on manufacturer's 301127.1 Rev. D manual, pages iiand 9 through 12. Use pH 4.01 and pH 7.00 buffers to calibrate the pHmeter. Total Plate Determined as per US Pharmacopoeia XXX, NF25, <61>,CFU/ Count Microbiological Limit Tests gm Mold/Yeast Determined as perUS Pharmacopoeia XXX, NF25, <61>, CFU/ Microbiological Limit Tests gm E.coli Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU/Microbiological Limit Tests gm Salmonella sp. Determined as per USPharmacopoeia XXX, NF25, <61>, CFU/ Microbiological Limit Tests gmStaphylococcus Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU/aureus Microbiological Limit Tests gm Pseudomonas Determined as per USPharmacopoeia XXX, NF25, <61>, CFU/ sp. Microbiological Limit Tests gmBenzophenones, Dal Molin MM, Silva S, Alves DR, Quintão NLM, Delle %Monache F et al.(2012) Phytochemical analysis and antinociceptiveproperties of Garcinia achachariu Rusby (Clusiaceae) seeds. Arch PharmRes 35: 623-631. Protein Amino acid analysis conducted on Hitachi L-8900amino acid % analyzer according the manufacturer's instructions.

Some physico-chemical characteristics of the present achacha serumfractions and extracts are illustrated in Tables 6, 7, and 8 below. Theserum fractions and extracts are prepared according to the methodsdescribed in the corresponding example(s) above. The achacha serumfraction included in the finished ingredient Recentia® GH-P (CASRN#1622986-60-0), which is illustrated in Table 8 below, is preparedaccording to the process described in Example 2.

TABLE 6 Serum Fractions Serums obtained from: Fruit Whole Fruit FruitPeel Flesh and Seed Lot GH 0785 Lot GH 1082 Lot GH 1083 Appearance ClearOrange Clear Orange Hazy Yellow Liquid Liquid Liquid Odor CharacteristicCharacteristic Characteristic Color (Gardner Scale) 6.5 8.5 6.5 Drymatter (%) 13.05 11.02 16.3 Refractive index (nD) 1.355 1.352 1.359 pH3.06 2.85 4.03 Density, g/cm³ 1.0612 1.0514 1.0714 Protein <0.07% <0.13%<0.15% Benzophenones  <0.1%  <0.1%  <0.1%

TABLE 7 Extracts Extracts (1 part plant material +3 part solvent ratio,weight/weight) obtained from: Fruit Peel Fruit Peel Whole FruitExtracted at Whole Fruit Extracted at Extracted at 4° C. Extracted at40° C. 4° C. Lot GH (PE) 40° C. Lot GH (PE) Lot. GH 0841 0837 Lot. GH0842 0838 Appearance Orange Orange Orange Liquid Orange Liquid LiquidLiquid Odor Characteristic Characteristic Characteristic CharacteristicColor (Gardner Scale) 11.0 12.5 11.5 13.5 Refractive index (nD) 1.43151.4291 1.4314 1.4291 Protein <0.046% <0.049% <0.041% <0.046%Benzophenones About 0.1%  <0.1% About 0.1% or  <0.1% higher

TABLE 8 Recentia ® GH-P Test Parameter Result Appearance Clear OrangeLiquid Odor Characteristic Solubility in water Soluble in any ratioColor (Gardner scale)  5-12 Dry matter (%)  9.0-12.1 pH 2.9-3.8Refractive index (nD) 1.349-1.355 Total Plate Count (CFU/g) <100Mold/Yeast (CFU/g) <100 E. coli (CFU/g) Negative/10 g Salmonella sp.(CFU/g) Negative/10 g Staphylococcus aureus (CFU/g) Negative/10 gPseudomonas sp. (CFU/g) Negative/10 g

Example 8—Absorbance Spectra

Absorbance spectra (in wavelength ranges 200-400 nm and 400-1000 nm)were obtained from 100 microliter samples of serum fractions andextracts of achachariu using a Synergy 2 multi-mode microplate reader(BioTek Instruments, Inc) with 96-well black quartz microplate (HellmaAnalytics GmbH). All dilutions were done as volume/volume. Spectra of100 microliter aliquots of respective solvents (ultrapure deionizedwater and fragrance grade dipropylene glycol) were subtracted fromsample spectra. A summary of the absorbance spectra results areillustrated in FIGS. 3A, 3B, 4A, and 4B. FIGS. 3A and 4A illustrateabsorbance spectra of a wavelength range of 200-400 nm. The samples ofachacha serum fractions and extracts analyzed in FIGS. 3A and 3Bincluded GH 0785, GH 1082, and GH 1083. The samples of achacha serumfractions and extracts analyzed in FIGS. 4A and 4B included GH 0837, GH0838, GH 0841, and GH 0842.

Example 9—Biological Activity

Serums and finished ingredients prepared according to variousembodiments herein were evaluated for biological activity of interest.The evaluations included in this example are: cytotoxicity; skinsensitization potential; inhibition of trypsin activity; inhibition ofelastase activity; inhibition of Kallikrein 5; inhibition of IL-6, IL-8,and/or PGE2; ARE activation; melanin synthesis inhibition; lipogenesisinhibition; and protein loss inhibition in hair. The test methods andresults of these evaluations are discussed in more detail below.

Evaluation of Cytotoxicity

Lactate Dehydrogenase (LDH) is a key cytoplasmic enzyme. Presence of LDHoutside the cells at levels above normal background leakage is anindicator of cell damage or cell death. Assays quantifying LDH in cellculture medium are commonly employed to assess potential cytotoxicity.Observation of cells under a microscope that identify cell rupture orchanges in cell morphology can also contribute to the assessment ofcytotoxicity.

Normal human adult epidermal keratinocytes (HEK) and all cell culturesupplies in the cytotoxicity evaluation were obtained from LifeTechnologies Co. (Carlsbad, Calif., USA). The cells were grown and thenmaintained in keratinocyte basal medium 154 (M154) with added humankeratinocyte growth supplements (HKGS) at 37° C. in an atmosphere of 5%CO₂ and used between passages 2 to 4. For the experiments, HEK cellswere trypsinized, seeded in 96-well plates, and grown to ˜80%confluence. HEK were then exposed, or not, to a stress factor, andincubated for 16 hours with or without test articles at variousconcentrations. After incubation, HEK cell supernatant medium sampleswere collected, and levels of LDH were evaluated using Cytoscan™ LDHAssay kit (Catalog #786-210, produced by G-Biosciences, St. Louis, Mo.,USA). Untreated, unstressed HEK cells were lysed using kit-suppliedlysis buffer as a positive control, with lysate used as assay positivecontrol and a measure of maximum LDH release. Lower induced LDH release,when confirmed by microscopy, indicates lower cytotoxicity.

Evaluation of Skin Sensitization Potential

It is possible for a material to not be cytotoxic, and yet be unsafe dueto provoking an allergic reaction upon skin contact. Typically, initialexposure to an allergen sensitizes the immune system, and followingexposures cause an allergic response. Recent progress in understandingthe mechanisms of skin sensitization identified interleukin-18 (IL-18)production in normal human epidermal keratinocytes (HEK) as a usefulbiomarker for skin contact sensitization (Corsini, et al., “Use of IL-18Production In a Human Keratinocyte Cell Line to Discriminate ContactSensitizers from Irritants and Low Molecular Weight RespiratoryAllergens.” Toxicol In Vitro. 2009 August; 23(5):789-96; Teunis, et al.,“Transfer of a Two-tiered Keratinocyte Assay: IL-18 Production byNCTC2544 to Determine the Skin Sensitizing Capacity and EpidermalEquivalent Assay to Determine Sensitizer Potency.” Toxicol In Vitro.2013 April; 27(3):1135-50). IL-18 is considered a suitable in vitroalternative to animal skin sensitization testing methods such as theLocal Lymph Node Assay. Thus, IL-18 production in HEK was evaluated todetermine sensitization potential.

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. After incubation withtest articles or controls for 16 hours, the HEK cells were lysed with100 μl/well of 0.5% Triton X-100 in pH 7.4 Phosphate Buffered Saline(PBS). The cell lysates were collected, and IL-18 was quantified usingHuman IL-18 ELISA Kit (Catalog #7620, produced by MBL International Co.,Woburn, Mass., USA). A known skin sensitizer, para-phenylenediamine(pPD) used as a positive control, significantly induced IL-18 comparedto vehicle control in HEK cultures. Fold changes of IL-18 levels betweentest articles and respective vehicle controls were calculated andcompared to pPD (positive control). Lower induction of IL-18 indicateslower sensitization potential.

Inhibition of Trypsin Activity

Collagen fibers provide mechanical strength and support for the skin. Aubiquitous protease, trypsin, is associated with damage andinflammation. Trypsin breaks down collagen, potentially leading todecreased mechanical strength of the skin, as well as wrinkles anddarkening after stress or injury (Burns T, Breathnach S, Cox N,Griffiths C. Rook's Textbook of Dermatology. Eighth Edition.Wiley-Blackwell, 2010. Vol. 1 Sections 8.21 to 8.27. Vol. 2 Section29.7).

Trypsin inhibition was determined via an EnzChek kit utilizing caseinsubstrate with intra-molecularly quenched fluorescent label moieties(Catalog # E6638, produced by Life Technologies). Testing was conductedaccording to manufacturer instructions. Digestion buffer concentrate wasdiluted in deionized water. Substrate and bovine trypsin (Sigma catalognumber T9201) were dissolved and diluted in the digestion buffer. Testarticles were dissolved and diluted in digestion buffer. Calibrationcurve was constructed with amounts of trypsin ranging from 1000nanograms to about 1.4 nanograms in reaction volume. Soybean trypsininhibitor, type I-S(Sigma) was used as a positive control.

Amount of trypsin in wells with test articles and controls was fixed at1000 nanograms. IC₅₀ was calculated as concentration of test article inthe reaction volume (e.g. microtiter plate well) necessary to reduce thetrypsin activity to 50%. Lower IC₅₀ values indicate higher potency and adegree of efficacy.

Inhibition of Elastase Activity

Elastin is a protein essential to elastic fiber network contained inconnective tissues which depend on elasticity for their function, suchas skin. Excessive elastase activity, commonly related to inflammation,degrades elastin and decreases strength and resilience of the skin.During inflammatory processes, elastase can be found in areas beyondthose where it is produced or secreted. Human neutrophil elastaseinhibition by test articles was determined in kinetic colorimetric assaydescribed by Elastin Products Company, Inc. (Elastin Products Company.Assay with N-MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) as substrate.Elastin Products Company, Inc. Research Biochemicals Catalogue. 2004. p.84) and modified for its use with 96-well microtiter plates (Corning3641) from Corning, Inc. (and Synergy 2 microplate reader from BioTekInstruments, Inc. The N-Methoxysuccinyl-Ala-Ala-Pro-Val-pNA substrate(EPC, Catalog No: FH237), and elastase (EPC SE563) were from ElastinProducts Company (Owensville, Mich., USA). Working solution of elastasewas prepared with 0.15 M pH 7.5 Tris-HCl buffer containing 50 mM NaCl.Working solution of substrate was prepared in 0.15 M pH 5.0 acetatebuffer containing 100 mM NaCl, with an aliquot of 2% by volume of finalbuffer of 1-methyl-2-pyrrolidone used for initial dissolution of thesubstrate. Deionized water was used to dissolve buffer components.Reaction volume in each well was 224 μl; concentration of elastase was0.87 units/ml, and substrate, 363 μM.

Enzymatic activity in cleaving the substrate was indicated by adevelopment of yellow color measured as increase in absorbance at 410 nmwavelength. The mean of maximum rate of absorbance increase in negativecontrol wells was considered as 100% of enzyme activity. IC₅₀ wascalculated as concentration of test article in the well which reducedthe elastase activity to 50%. Lower IC₅₀ values indicate higher potencyand a degree of efficacy.

Inhibition of Kallikrein 5

Kallikrein 5 (KLKS), also known as stratum corneum tryptic enzyme, is atrypsin-like serine protease. Recent in vitro and in vivo evidenceimplicates increased levels of KLKS in augmented inflammatory responsesuch as rosacea (Two A M, Del Rosso J Q, Kallikrein 5-mediatedinflammation in rosacea: clinically relevant correlations with acute andchronic manifestations in rosacea and how individual treatments mayprovide therapeutic benefit. J Clin Aesthet Dermatol. 2014 January;7(1): 20-5) and in induction of atopic dermatitis-like lesions (Briot A.et al., Kallikrein 5 induces atopic dermatitis-like lesions throughPAR2-mediated thymic stromal lymphopoietin expression in Nethertonsyndrome. J Exp Med. 2009 May 11; 206(5):1135-47). Normal human adultepidermal keratinocytes (HEK) were cultured as described above for thecytoxicity evaluation. After incubation with test articles or controlsfor 16 hours, HEK cell culture supernatants were collected. KLKS wasquantified using a human KLKS immunoassay Quantikine ELISA kit (Catalog# DKK500, produced by R&D Systems, Minneapolis, Minn.). The changes ofKLKS concentrations between test articles and vehicle controls werecalculated and compared. IC₅₀ (concentration of test article necessaryto reduce KLKS levels to 50% compared to samples from untreated cells)values were calculated by sigmoidal curve fitting with SigmaPlot 10.0(Systat Software). Lower IC₅₀ values indicate higher potency and adegree of efficacy.

Inhibition of IL-6 and/or IL-8 Induced by SDS

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. The cells were thenincubated with test articles and/or controls for 16 hours. Presence ofsodium dodecyl sulfate (SDS) in cell cultivation medium at specificconcentrations was used for induction of chemokines and cytokines. IL-8was induced by 6 μg/mL SDS, IL-6 by 12.5 μg/mL SDS. After incubation,HEK cell supernatants were collected. Quantikine® ELISA kits (R&DSystems Inc, Minneapolis, Minn.) were used to quantify theseinterleukins in the supernatants. IL-8 was quantified by Human CXCL/IL-8Immunoassay kit (Catalog # D8000C), and IL-6 was quantified by HumanIL-6 Immunoassay kit (Catalog # D6050). IC₅₀ (concentration of testarticle necessary to reduce interleukin levels to 50%, with samples fromuntreated cells considered as 0% and samples treated solely withrespective inducing quantity of SDS as 100%) values were calculated bysigmoidal curve fitting with SigmaPlot 10.0 (Systat Software). LowerIC₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of IL-6, IL-8, and PGE2 Induced by Full-Spectrum Sunlightfrom Artificial Source

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. The cells were washedonce, and M154 was replaced with PBS. Both the washing and thereplacement were done with PBS to remove light-absorbing components ofM154. The 96-well plate containing HEK was then covered withUV-transparent 1 mm quartz sheet, placed on white underlay atopcontrolled Peltier-cooled surface maintaining room temperature, andirradiated with a dose of 20 J/cm² of artificially produced fullspectrum sunlight at dose rate of about 1100 W/m², as measured viapyranometer through same quartz cover. PBS was then removed and replacedwith M154, and cells were incubated with test articles and/or controlsfor 16 hours. Identical manipulations, with exception of presence ofsunlight, were carried out with HEK serving as unstressed controls.Irradiation equipment was obtained from Solar Light Company, Glenside,Pa. and included Solar Simulator LS1000-6R-002 in Airmass 1.5configuration using plain mirror; XPS1000 precision current source, andPMA2144 Pyranometer. After incubation, HEK cell supernatants werecollected. Quantikine® ELISA kits (R&D Systems Inc, Minneapolis, Minn.)were used to quantify interleukins in the supernatants. IL-8 wasquantified by Human CXCUIL-8 Immunoassay kit (Catalog # D8000C), IL-6was quantified by Human IL-6 Immunoassay kit (Catalog # D6050); and PGE2was quantified using Parameter™ Prostaglandin E₂ Assay (Catalog #KGE004B). IC₅₀ (concentration of test article necessary to reduceinterleukin or prostaglandin levels to 50%, with samples fromnon-irradiated cells considered as 0% and from irradiated cellsconsidered as 100%) values were calculated by sigmoidal curve fittingwith SigmaPlot 10.0 (Systat Software). Lower IC₅₀ values indicate higherpotency and a degree of efficacy.

Table 9 summarizes the results of evaluating inhibition of IL8 and PGE2activity induced by full-spectrum sunlight from an artificial source andcytotoxicity (LDH/microscopy).

TABLE 9 IL8/full spectrum PGE2/full spectrum Cytotoxicity Material suninduction sun induction (LDH/microscopy) Whole Fruit Some inhibition atInhibits at low Cytotoxic at 0.1% Extract, Lot 0841 low concentrationsconcentrations, IC₅₀ and above (~25% inhibition at 0.01% 0.01%) FruitPeel Extract, Inhibition at low Inhibits at low No cytotoxicity at Lot0837 concentrations concentrations, IC₅₀ 0.1% (estimated IC₅₀ 0.04%0.012%, max inhibition 66% at 0.05%) Fruit Peel Serum No significantIC₅₀ between No cytotoxicity at Fraction, Lot 1082 inhibition0.003-0.03%. Maximum 0.1% inhibition 70% at 0.001% Whole Fruit Serum Nosignificant Maximum inhibition No cytotoxicity at Fraction, Lot 0785inhibition 40% at 0.01% 0.1% Fruit Flesh + Fruit No significantNon-significant No cytotoxicity at Seed Serum inhibition inhibition at0.01-0.02% 0.1% Fraction, Lot 1083

Table 10 summarizes the results of evaluating inhibition of IL8 inducedby SDS; KLKS inhibition; IL18 inhibition; trypsin inhibition; elastaseinhibition; and cytotoxicity.

TABLE 10 IL8 Cytotoxicity inhibition (LDH/ Trypsin Elastase Material(SDS) KLK5 IL18 microscopy) Inhibition Inhibition Achachariu Not testedIC₅₀ Maximum No IC₅₀ IC₅₀ (Garcinia 0.18% 1.46 fold cytotoxicity 0.37%0.26% humilis) Fruit (benchmark at 0.1% Peel Serum sensitizer Fraction,7.8 fold) Lot 0822 Achachariu About 65% IC₅₀ Maximum No IC₅₀ IC₅₀(Garcinia inhibition  0.2% 1.8 fold cytotoxicity 0.32% 0.28% humilis) at0.1% (benchmark at 0.1% Whole Fruit sensitizer Serum 7.8 fold) Fraction,Lot 0786

In summary, biological activity results suggest that preparations ofachachariu: (i) are not skin sensitizers; (ii) are not cytotoxic atselected concentrations that could be relevant to their concentrationsin finished product formulation; and (iii) potential usefulness formitigating signs of skin aging caused by inflammation and relatedprocess (e.g., those triggered by stresses to the skin, includingenvironmental stress such as full-spectrum sun exposure). Morespecifically, the test results suggested the following: (i) againstPGE2, achachariu whole fruit extract is slightly more effective thanfruit peel extract; (ii) against PGE2, achachariu fruit peel serumfraction is more effective than whole fruit serum fraction, which istentatively better than fruit flesh+ fruit seed serum fraction; and(iii) achachariu whole fruit serum fraction is effective for inhibitingSDS-induced IL-8 activity, but not against sun-induced IL8, which issurprising. Also surprising was: (i) the potent inhibition demonstratedby fruit peel extract against sun-induced IL8 and fruit peel serumfraction against sun-induced PGE2; (ii) the lack of any notable activityby fruit flesh+fruit seed serum fraction; (iii) fruit peel serumfraction and whole fruit serum fraction are more cytotoxic than fruitflesh+fruit seed serum fraction; and (iv) whole fruit extract is morecytotoxic than fruit peel extract.

Anti-Oxidant Response Element Activation

The ARE is the so-called “master switch” believed to control theantioxidant defense system of most cells. When the ARE is activated inresponse to oxidative stress, the corresponding genes signal the cell tobegin producing reduction/oxidation regulators and/or reactive oxygenspecies (“ROS”) quenching proteins and enzymes. ROS are highly reactivemolecules formed naturally within cells as a natural byproduct of thenormal metabolism of oxygen and play a role in cell signaling andhomeostasis. However, when a cell is exposed to a stressor such as heator UV radiation, ROS levels can increase, and in some instancesdramatically. As the damage caused by ROS accumulates over time, itcauses more and more oxidative stress at the cellular level thatultimately may lead to tissue damage and/or organ dysfunction. Thus,without being limited by theory, it is believed that if achachariu candemonstrate the ability to activate the ARE, then applying an effectiveamount of achachariu to keratinous tissue may help fight cellular damageassociated with oxidative stress.

ARE activation was quantitated using the ARE-32 reporter cell lineavailable from CXR-Biosciences as described below. ARE-32 is a stableMCF7 cell line containing pGL8x-ARE (8 copies of the rat GST ARE linkedto the luciferase gene) and pCDNA3.1, which contains the neomycinselectable marker. Selection was performed in the presence of G418 andresistant clones were isolated. Clones were screened for induction ofluciferase in response to tBHQ.

The ARE-32 cells are maintained routinely in Dulbecco's Modified EagleMedium (phenol red free) (“DMEM”) containing: 10% fetal bovine serum(“FBS”), 50 units/ml penicillin & 50 μg/ml streptomycin, 0.8 mg/ml G418.Cells are subcultured every 3-4 days. If needed, cells can be frozen inmedium that contains 90% FBS and 10% DMSO.

ARE Method

In a 96 well-plate, 1×10⁴ cells/well are seeded in 100 μl DMEMcontaining 50 units/ml penicillin, 50 μg/ml streptomycin, 0.8 mg/ml G418and 10% FBS. Next, the cells are incubated at 37° C. in a 5% CO₂incubator for 24 hrs, and then the medium is replaced with 100 μl freshmedia. The test samples are treated with achachariu serum fractions atthe concentration listed in Table 11 (1 ul per well), the positivecontrol is 25 uM TBHQ. (10 mM tBHQ of stock solution freshly prepared inDMSO). 100 ul of media is added after treatment for a final assay volumeof 200 uL. The cells are incubated at 37° C. in CO₂ incubator foranother 24 hrs. The test samples are then assayed for luciferaseactivity with Steady-Glo™ brand assay system according to themanufacturer's instruction.

The results of the test are summarized in Table 11. At concentrations of0.167% to 1.5%, both the whole fruit and peel serum fractions appear toprovide more ARE activation than than the control. The whole fruit alsodemonstrated a directional increase at 0.056% versus the control.

TABLE 11 ARE Activation Whole fruit¹ Peel² % increase p-value vs. %increase p-value vs. v/v % achachariu vs control control vs controlcontrol 1.5 929 0.000087 1133 0.000046 0.5 489 0.0023 614 0.00086 0.167197 0.0076 209 0.0043 0.056 134 .0.091 113 0.67 0.0185 103 0.96 92 0.790.006173 112 0.83 99 0.99 0.002058 93 0.89 92 0.94 0.000686 120 0.78 930.94 ¹Recentia ® GH from AkzoNobel ²Recentia ® GH-P from AkzoNobel

Melanin Synthesis Inhibition—B16 Assay

Overproduction of melanin is generally associated with a variety of skinpigmentation conditions (e.g., age spots, vitiligo, solar lentigines,and melasma). Thus, without being limited by theory, it is believed thatif achachariu can demonstrate the ability to inhibit melanin production,then applying an effective amount of achachariu to skin may help improvethe appearance of skin pigmentation conditions.

A commercially available B16-F1 mouse melanoma cell line from AmericanTissue Culture Collection, Virginia, USA was employed in a conventionalmelanin synthesis inhibition assay. The cell culture medium used in theassay is 500 mL of Dulbecco's Modified Eagle's Medium (“DMEM”), 50 mLFetal Bovine Serum (“FBS”), and 5 mL of penicillin-streptomycin liquid.B16-F1 cells that are cultured in this medium and grown to greater than90% confluency will synthesize melanin. While not intending to be boundby theory, it is hypothesized that melanin synthesis is stimulated bythe culture medium and/or stress induced by growth to a high confluency.The DMEM and FBS can be obtained from American Tissue Culture Collectionand the penicillin-streptomycin liquid can be obtained from Invitrogen,Inc., California, USA. Equipment used in the assay include a CO₂incubator (e.g., a Forma Series Model 3110 by Therma Scientific,Massachusetts, USA or equivalent); a Hemocytometer (e.g., Bright Linemodel by Hauser Scientific, Pennsylvania, USA or equivalent); and aUV-Visible Spectrum Plate Reader (e.g., SpectraMax250 from MolecularDevices, California, USA or equivalent).

Day 0: To begin the assay, the cell culture medium is heated to 37° C.and 29 mL of the medium is placed into a T-150 flask. Approximately1×10⁶ of B16-F1 passage 1 mouse cells are added to the T-150 flask andincubated for 3 days at 37° C., 5% CO₂, 90% relative humidity, until˜80% confluency.

Day 3: The cells from the T-150 flask are trypsinized, and the number ofcells is determined using the Hemocytometer. Initiate a 96 well platewith 2,500 cells per well in 100 μL of cell culture medium. Incubate theplate at 37° C., 5% CO₂, 90% relative humidity for 2 days until at least20% to 40% confluent.

Day 5: Remove the cell culture medium from the plate and replace withfresh culture medium (100 uL per well). Add 1 uL of test compounddiluted in a water solvent. Multiple dilution ratios may be tested inorder to generate a dose response curve, wherein preferably three wellsare treated with each dilution ratio. Positive and negative controls mayinclude wells having the cell culture medium, B16-F1 cells, and thesolvent (negative control), and wells comprising the cell culturemedium, B16-F1 cells and and a known melanin inhibitor (e.g.,deoxyarbutin or kojic acid).

Day 7: Cells should have greater than ˜90% confluency. If not, this datapoint is not used. Add 100 uL of a 0.75% sodium hydroxide solution toeach well. Read the 96-well plate using the UV-Vis Plate Reader at 410nm to optically measure the amount of melanin produced between wellsthat are treated with the fava bean extract and control wells that arenot. Wells in which melanin is produced appear brownish in color. Wellsin which little melanin is produced appear clear to light purple incolor. Percentage of melanin synthesis inhibition is calculated by thefollowing equation:

$\frac{100 - {\left\lbrack {{{OD}\; 410\mspace{14mu} {Test}\mspace{14mu} {Compound}} - {{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 2}}} \right\rbrack \times 100}}{\left( {{{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 1}} - {{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 2}}} \right)}$

Where OD410 is the Optical Density at 410 nm as measured by the UV-VisSpectrum Plate Reader.

When Control #3 is used, the formula for percentage melanin synthesisinhibition is:

$\frac{100 - {\left\lbrack {{{OD}\; 410\mspace{14mu} {Test}\mspace{14mu} {Compound}} - {{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 3}}} \right\rbrack \times 100}}{\left( {{{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 1}} - {{OD}\; 410\mspace{14mu} {Control}\mspace{14mu} {\# 2}}} \right)}$

The concentration of test agent needed to provide the IC 50 is recorded.

The results of the test are summarized in Table 12, which shows thatachachariu inhibits melanin synthesis, and thus is expected to provide askin lightening benefit.

TABLE 12 Melanin Synthesis Inhibition B16 (IC 50) Concentration Neededfor IC 50 Composition (v/v %) Achachariu¹ (whole fruit) 0.19 Achachariu²(peel only) 0.19 ¹Recentia ® GH from AkzoNobel ²Recentia ® GH-P fromAkzoNobel

Lipogenesis Inhibition

This example demonstrates the ability of achachariu to inhibitlipogenesis in human pre-adipocytes. Lipogeneis involves the synthesisof commonly known lipids such as fatty acids and triglycerides, and isone of primary ways mammals store energy. However, lipogenesis alsoinvolves the synthesis of lipids such as sebum. Sebum is a lipidproduced by sebocytes, which are a type of skin cell found primarily inthe sebaceous glands of mammalian skin. Sebum is produced by the body tolubricate and waterproof the skin and hair of mammals. However,overproduction of sebum can result in oily appearing skin and/or skinthat appears to have poor texture. Thus, without being limited bytheory, it is believed that if achachariu can demonstrate the ability toinhibit lipogenesis, then applying an effective amount of achachariu tokeratinous tissue may help regulate conditions associated with sebumoverproduction.

Method

Human pre-adipocytes were selected for use in this example. Because ofthe known difficulty associated with culturing and testing sebocytes,pre-adipocytes are commonly used as a surrogate for sebocytes todetermine the potential of a test agent to inhibit sebum production.

Human subcutaneous pre-adipocytes purchased from Zen-Bio, Inc (Cat. #SP-F-SL) were cultured in PM-1 media (available from Zen-Bio, Inc asCat# PM-1 (plus 5 ng/ml EGF)) to 80-90% confluency. The cells weretransferred to 96-well clear bottom white plates to provideapproximately 40,625 cells/cm² in the well (˜12,500 cells) and 150 μl ofPM-1 media per well, and then cultured for 24-48 hours in a 5% CO₂incubator at 37° C. The PM-1 media was then replaced withdifferentiation medium (Zen-Bio, Inc. Cat# DM-1), and the cells wereincubated for another 6 days. After incubating in the differentiationmedium, 90 μl of the differentiation medium was carefully replaced with140 μl of human subcutaneous adipocyte medium ((Zen-Bio, Inc. Cat#AM-1). Care was taken not to touch or disturb the cells at the bottom ofthe well. 2 μl of achachariu (Recentia® GH-P for AkzoNoble) or controlcomposition (100 μM Genistein (Cat# G6649) from Sigma) was added to eachwell daily for 9 days (total incubation of 15 days). On Day 15, 5 μL ofAdipoRed reagent (Lonza; Cat. Number: PT-7009) was slowly added directlyto cells in the treatment medium, and the plate was gently mixed aftereach row addition. The plate was incubated for 15 minutes at roomtemperature. Lipogenesis was quantitated using an EnVision® brandFluorescent spectrophotometer Plate Reader according to the AdipoRedprotocol. The plates were scanned from the bottom using the 451 mirrorand (excitation 485 nm; emission 535) filter. Each well was scanned in aZ pattern (7 reads across from left to right, 7 reads diagonally fromright to left and 7 reads across from left to right for a total of 21end points).

Percent inhibition was calculated as:

Average Control RFU−Sample RFU×100/Average Control RFU

The cells were assayed and normalized to the control by using aFluoReporter® Blue Fluorometric brand dsDNA Quantitation Kit.Immediately after the screen the AdipoRed containing cell media wasgently aspirated, cells were rinsed with 100 μl 1×PBS taking care not todislodge them from the bottom and 100 μl distilled water was added/well.The plates were frozen at −80° C. to lyse the cells and assayedaccording to the kit instructions at a later date.

The results of the test are summarized in Table 13, which shows thatachachariu inhibits lipogenesis, and thus is expected to help regulateconditions associated with the overproduction of sebum.

TABLE 13 Lipogenesis Inhibition Compostion IC 50 w/v % Recentia ® GH-P(achachariu peel) 0.4% Recentia ® GH (achachariu whole fruit) 0.5%

Inhibiting the Cell's Inflammation Response to a Stressor NF-Kappa-Beta(“NF-kB”) Assay.

NF-kB (i.e., nuclear factor kappa-light-chain-enhancer of activated Bcells) is a protein complex that belongs to the category of“rapid-acting” primary transcription factors transcription factors thatare present in cells in an inactive state and do not require new proteinsynthesis in order to become activated), which allows NF-kB to be afirst responder to harmful cellular stimuli such as ROS and otherstressors. NF-kB is found in almost all animal cell types and is knownto be involved in the cellular inflammation pathway. Cellularinflammation is associated with a variety of skin conditions, and thusinhibiting NF-kB activation vis-à-vis cellular inflammation with aneffective amount of achachariu may help treat these types of skinconditions.

Method

CellSensor™ NF-κB-bla HEK 293T cells (Invitrogen, Cat. # K1165) wereplated in assay medium (DMEM with high glucose (Gibco, Cat. #11965) plus10% dialyzed FBS). The cells were cultured and seeded at 10,000/well in96-well plates (black-sided Poly-D-Lysine coated plates, BD #356692),and then incubated at 37° C. and 95% RH for the 72 hours prior totesting. Recombinant human TNFα (available from R&D systems) was used tostimulate NF-kB activation in the cells. Fisetin (3, 7, 3′,4′-tetrahydroxyflavone) was used to inhibit stimulation of the cells byTNFα. A ToxBLAzer™ DualScreen brand screening kit (Invitrogen, Cat.#K1138) was used as the fluorescent substrate according to themanufacturer's instructions.

The following controls were run on each plate in the assay:

High control(Stimulated)=cells+TNFα+1% DMSO

Blank(Unstimulated)=cells+1% DMSO

Standard(Positive control inhibitor)=cells+TNFα+Fisetin+1% DMSO

Negative Control(no cells)=assay medium

NF-κB % inhibition is calculated as:

$\frac{{{High}\mspace{14mu} {Control}} - {Sample}}{{{High}\mspace{14mu} {Control}} - {Blank}} \times 100$

The results of this test are summarized in Table 14 below. Asillustrated in Table 14, the achachariu whole fruit serum fractions andachachariu peel serum fractions provided an IC 50 for NF-kB inhibitionof 2%. Thus, applying an effective amount of achachariu to a targetportion of keratinous tissue may help regulate conditions related tocellular inflammation.

TABLE 14 IC Compostion 50 (v/v %) Cytotoxicity Recentia ® GH-P(Achachariu Peel only) 2% None observed Recentia ® GH (Achachariu wholefruit) 2% None observed

Inhibiting the Cell's Inflammation Response to a Stressor—ProstaglandinE2 (“PGE2”) Assay.

PGE2 is a hormone-like substance that is known to participate inmodulation of inflammation. Cellular inflammation is associated with avariety of skin conditions, and thus inhibiting PGE2 activationvis-à-vis cellular inflammation may help treat these types of skinconditions.

Method

Tert keratinocytes (“tKC”) were plated at 40,000 cells/well into 24-wellplates in 1 ml/well volume. EpiLife Medium (Life Technologies cat #MEPICFPRF500) supplemented with keratinocyte growth supplement (Lifetechnologies cat #S-001-5) was used as the assay media. The cells weregrown to confluence/near confluence, and then subjected to 15 mJ/cm²UVB-stress. The test compositions (achachariu and vehicle control)(diluted 1:1000) were added, and the plates were incubated for 18-24hours. The supernatant was removed from each well, and the cells wererinsed with 2 ml/well medium (without supplements). A Cell Titer-Gloassay (measures ATP activity) was conducted on the cells fornormalization. The supernatant was tested in a PGE2 assay (ProstaglandinE2 Assay kit from Cisbio Bioassays cat#62P2APEB) according to themanufacturer's instructions. The PGE2 results were normalized to ATPactivity.

The results of the PGE2 assay are summarized in Table 15. The achachariuwhole fruit serum fraction and achachariu peel serum fractions bothdemonstrated the ability to inhibit release of PGE2 from keratinocytesexposed to 15 mJ/cm2 UVB radiation, which illustrates anti-inflammatoryactivity of achachariu. Thus, a personal care composition comprisingachachariu may be useful for regulating a condition of mammaliankeratinous tissue related to cellular inflammation.

TABLE 15 PGE2 Release (% of vehicle Test control) StDev Recentia ® GH-PAchachariu peel 28% 8.20% Recentia ® GH (Achachariu whole fruit) 52%11.40% Vehicle control 98% 13.20%

Protein Loss Inhibition in Hair Hair is a protein filament formedprimarily of keratin that grows from follicles found in the dermis ofmammalian skin. The proteins (i.e., keratin) in hair can be damaged by avariety of endogenous and exogenous stressors (e.g., UV radiation),which can lead to, e.g., dry, brittle, and/or dull looking hair. Thus,it would be desirable to inhibit, prevent or even reverse undesirableprotein loss in hair.

It is believed, without being limited by theory, that changes in thelevel of certain amino acids found in keratin correspond to the overallchange in protein level in hair. In this test, serine, proline, glutamicacid, and valine were used as surrogates for overall protein level,since these amino acid residues represent approximately 40% of theprotein concentration.

Method.

Achachariu whole fruit serum extract (Recentia® GH) was formulated at 2%(w/v) in a 50:50 ethanol:water chassis with 2% SEPIMAX ZEN brandthickener (Seppic, France) to a final pH of 6.0 to provide a testcomposition. The same composition, except without any achachariu serumfraction, was used as a control. The test composition and controlcomposition were applied to light-brown, non-chemically-treated, 2 g,flat hair switches (6 switches per leg) at 0.2 g/g, and thoroughlydistributed through-out the hair switch. Twenty-four hours aftertreatment, the switches were washed with Pantene® Volume brand shampoo(from the Procter & Gamble Co.). This treatment/wash cycle was repeatedten times, but the hair switches were not washed after the tenthtreatment. After the tenth treatment, half the switches from the controland test group were tested for protein loss (0 hours), and the otherhalf were exposed to 40 hours of ultraviolet radiation in an Atlas™Ci3000+ brand weather-o-meter and then tested for protein loss (40hours). An internal and outer quartz filter was used to simulatebroad-spectrum, outdoor daylight with a specific irradiance of 1.48 W/m²at 420 nm. Temperature and relative humidity were kept constant at 35°C. and 80% RH. After the UV exposure, the hair switches were tested forprotein loss.

Protein levels are determined using the following method. 0.2-0.3 g hairsamples (2 inches length) are collected from each hair switch and areadded to glass scintillation vials. Distilled water is added at a ratioof 10:1 (mL water to g hair). Samples are shaken for 1 h at 2500 rpm ona DVX-2500 Multi-tube Vortexer platform. Amino acid concentration isdetermined after acid hydrolysis using High-Performance LiquidChromatography/Mass Spectrometry (HPLC/MS/MS). The analytes (Serine,Proline, Valine, Glutamic acid) and their corresponding internalstandards are subjected to hydrophilic interaction chromatography(HILIC) analysis on a ZIC-HILIC column (2.1×150 mm, 5 micron particles).Detection and quantitation is by tandem mass spectrometry operatingunder multiple reaction monitoring (MRM) MS/MS conditions. The aminoacid concentrations are determined by back-calculation using a weightedquadratic regression of a calibration curve generated from neatstandards.

The results of the test are summarized in Table 16 and illustrated inFIGS. 5 to 9. Amino acid levels are shown in μg/g. P-values of 0.1 orless are considered statistically significant. P-values of greater than0.1 but less than 0.2 are considered directional. As illustrated inTable 17 and FIGS. 5 to 9, the amount of amino acid loss observed whenthe samples were exposed to UV radiation for 40 hours increased relativeto the corresponding 0 hour samples. However, the achachariu-treatedsamples exhibited less protein loss at 0 hours and after 40 hours of UVexposure compared to their respective non-achachariu treatedcounterparts. In addition, the samples treated with achachariu did notexhibit a statistically significant difference in protein loss after 40hours of UV exposure when compared to the untreated samples at 0 hours.Thus, treatment with achachariu appears to reduce UV-induced proteinloss in hair, and the reduction in protein loss at 0 hours suggests thattreatment with achachariu may also reduce protein loss in hair due tonon-UV stressors.

TABLE 17 Serine Glutamine Valine Proline Total (a) 0 hours 32.00 24.588.50 14.48 79.54 Control (b) 0 hours 26.61 20.75 7.26 12.91 67.49treated w/ achachariu (c) 40 hours 41.87 35.05 12.82 21.03 110.79Control (d) 40 hours 31.38 25.26 8.90 14.95 80.48 treated w/ achachariuΔ_(a-b) ¹ 5.39 3.83 1.24 1.67 12.05 p-value 0.049 0.0646 0.1453 0.21650.0755 Δ_(a-c) ² 9.87 10.57 4.32 6.45 31.25 p-value 0.0007 <0.0001<0.0001 0.0001 <0.0001 Δ_(a-d) ³ 0.62 0.78 0.40 0.37 0.94 p-value 0.81900.6920 0.6412 0.7824 0.8899 Δ_(b-d) ⁴ 4.77 4.51 1.64 2.04 12.99 p-value0.062 0.0197 0.0446 0.1119 0.0442 ¹Difference between Control level at 0hours and Treated level at 0 hours. ²Difference between Control level at0 hours and Control level at 40 hours. ³Difference between Control levelat 0 hours and Treated level at 40 hours ⁴Difference between Treatedlevel at 40 hours and Treated level at 0 hours.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm”.

All documents cited in the Detailed Description of the Invention are, inrelevant part, incorporated herein by reference; the citation of anydocument is not to be construed as an admission that it is prior artwith respect to the present invention. To the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

Whereas particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of regulating a condition of mammaliankeratinous tissue, comprising: a. identifying a target portion ofkeratinous tissue in need of treatment or where treatment is desired;and b. applying a personal care composition comprising an effectiveamount of an achachariu serum fraction to the target portion ofkeratinous tissue during a treatment period, wherein the treatmentperiod is sufficient for the achachariu serum fraction to provide abenefit to the treated keratinous tissue.
 2. The method of claim 1,wherein the achachariu serum fraction is obtained from a peel of theachachariu fruit.
 3. The method of claim 1, wherein the achachariu serumfraction has a dry matter content of less than 25%.
 4. The method ofclaim 1, wherein the serum fraction is substantially free of at leastone of benzophenone and protein.
 5. The method of claim 1, wherein thepersonal care composition is applied to the target portion of keratinoustissue at least once per day.
 6. The method of claim 1, wherein thepersonal care composition is a rinse-off type composition, and at leastsome of the personal care composition is removed from the target portionof keratinous tissue after application.
 7. The method of claim 6,further comprising removing at least some of the personal carecomposition from the target portion of keratinous tissue with water. 8.The method of claim 6, further comprising removing at least some of thepersonal care composition from the target portion of keratinous tissuewith an implement.
 9. The method of claim 1, wherein the achachariuserum fraction is present in the personal care composition at an amountof from about 0.01% to about 15%.
 10. The method of claim 1, wherein thepersonal care composition further comprises about 20% to about 99.99% ofa dermatologically acceptable carrier.
 11. The method of claim 1,wherein the personal care composition comprises a preservative selectedfrom pentylene glycol, tetrasodium EDTA, sodium metabisulfite, potassiumsorbate, sodium benzoate.
 12. A method of treating a condition ofmammalian keratinous tissue, comprising: a. identifying a target portionof keratinous tissue in need of treatment; and b. applying a personalcare composition comprising an effective amount of an achachariu extractto the target portion of keratinous tissue.
 13. The method of claim 12,wherein the achachariu extract comprises polar compounds extracted fromachachariu plant material using a polar solvent.
 14. The method of claim12, wherein the achachariu extract comprises non-polar compoundsextracted from achachariu plant material using a non-polar solvent. 15.The method of claim 12, wherein the achachariu extract is obtained froman achachariu fruit.
 16. The method of claim 15, wherein the achachariuextract is obtained from the achachariu fruit peel.
 17. The method ofclaim 12, wherein the personal care composition is a rinse-off typecomposition, and at least some of the personal care composition isremoved from the target portion of keratinous tissue after application.18. The method of claim 17, further comprising rinsing at least some ofthe personal care composition off the target portion of keratinoustissue with water.
 19. The method of claim 17, further comprisingremoving at least some of the personal care composition from the targetportion of keratinous tissue with an implement.
 20. The method of claim12, wherein the achachariu extract is present at an amount of from about0.01% to about 15%.